The goal of this application is to identify phospholipid biomarkers of environmentally-induced (rotenone- induced) mitochondrial dysfunction associated with Parkinson's disease PD. This team has demonstrated that a mitochondria specific phospholipid, cardiolipin (CL), undergoes selective oxidation catalyzed by cytochrome c (cyt c) early during neuronal apoptosis. The central hypothesis is that exposure to the pesticide, rotenone causes time- and dose-dependent selective oxidation of CL and accumulation of its oxidized molecular species associated with mitochondrial dysfunction through enzymatic cyt c catalyzed reactions triggered early in apoptosis. The unique profile of CL molecular species represents a new type of biomarkers of rotenone-induced mitochondrial dysfunction associated with PD. Using oxidative lipidomics approach this group of investigators will first identify specific patterns of CL oxidized molecular species induced in rat primary cortical and midbrain neurons as well as neuroblastoma SH-SY5Y by rotenone. Further, the intent is to reveal the specific profiles of oxidized CL species in dopaminergic and cortical neurons using rat rotenone- infusion model of PD. Finally, some establishment for the presence of rotenone-specific CL oxidation patterns in human peripheral blood lymphocytes exposed to rotenone will be compared with those detected in the rotenone-infusion rat model. The following Specific Aims were developed to test the hypothesis: Specific Aim 1 will utilize oxidative lipidomics to identify and characterize molecular species of CL as well as unique stereo-specific oxygenated products of CL in primary rat cortical neurons and midbrain neurons as well as in SH-SY5Y cells upon exposure to rotenone. Specific Aim 2 will establish the mechanisms and pathways through which interactions of cyt c with CL are involved in CL oxidation in primary rat cortical and midbrain neurons as well as SH-SY5Y cells exposed to rotenone. Specific Aim 3 will determine the extent to which molecular species of rotenone-induced peroxidized CL detected and identified in neurons in vitro accumulate in mitochondria of cortical and midbrain neurons in vivo after infusion of rotenone to rats. Specific Aim 4 will reveal rotenone-specific CL peroxidation patterns in human peripheral blood lymphocytes as biomarkers of mitochondrial dysfunction associated with PD. PUBLIC HEALTH RELEVANCE: The goal of this application is to identify biomarkers of environmentally (rotenone)-induced mitochondrial dysfunction associated with Parkinson's Disease. This will be achieved by a novel oxidative lipidomics approach. Rotenone-specific peroxidation patterns of mitochondrial cardiolipins will be identified in rat cortical and midbrain neurons in vitro and in vivo and revealed in human peripheral blood lymphocytes.